Method of detecting and characterizing a nucleic acid or reactant for the application of this method

ABSTRACT

Method for detecting the possible presence of a DNA fragment, notably of a gene, in the midst of a complex sample of nucleic acids. 
     It comprises the hybridization of the sought fragment with a RNA probe, this being, prior or subsequent to the hybridization reaction, modified by an enzyme. 
     Application to seeking of particular genes or DNA fragments in the midst of a biological sample.

This application is a continuation of application Ser. No. 07/826,631filed Jan. 23, 1992, now abandoned, which is a continuation of U.S.application Ser. No. 07/715,854, filed Jun. 17, 1991, now abandoned,which is a continuation of U.S. application Ser. No. 07/513,040, filedApr. 23, 1990, now abandoned, which is a continuation of U.S.application Ser. No. 07/353,177, filed May 16, 1989, now abandoned,which is a continuation of U.S. application Ser. No. 06/848,239, filedApr. 4, 1986, now abandoned, which is a divisional of U.S. applicationSer. No. 06/373,017, filed Apr. 29, 1982, issued as U.S. Pat. No.4,581,333, which is a continuation of U.S. application Ser. No. 169,370,filed Jul. 16, 1980, now abandoned, which is a continuation of U.S.application Ser. No. 029,735, filed Apr. 13, 1979, now abandoned.

The invention relates to a method for detecting the presence and, ifnecessary, characterizing a nucleic acid or a sequence of the latter ina specimen which can contain it. It relates also to the reactantsnecessary for the application of this method. Finally it relates also tothe application of such a method, among other possible applications, tothe hybrid in vitro diagnosis of the presence in a biological specimen,derived notably from a human or animal host, of problem nucleicparticles, for example infectious in nature, or again the integrity ornot of this or that particular gene belonging to the normal geneticpatrimony of the host.

It is not necessary to dwell on the extraordinary richness in variousnucleic acids which any biological specimen can contain, for exampleblood, which it is possible to sample from any living creature. It isalso the same regarding different sequences, for example, of numerousgenes which any particular nucleic acid may contain in this specimen;whence the immense difficulties that the genetician may encounter at thelevel of the detection or characterization of certain nucleic acids in aspecimen, difficulties which also arise as soon as there is a questionof characterizing the presence of certain fragments, for example ofgenes, contained in these nucleic acids.

The characterization of a particular nucleic acid or of particulargenes--for example for the study of the organization of geneticsequences of DNA which contains them--hence involves the productionpreviously from the medium studied, of a fraction enriched in thisnucleic acid. To this end, there have already been proposed enrichmenttechniques exploiting hybridation reactions between the nucleic acid orthe gene sought and a probe, to the extent that the latter was availableand when the hybrids formed could then be separated from the medium, forexample by differential sedimentation in a solution subjected toultra-centrifugation.

Such probes have already been described: they are generally constitutedby ribonucleic acids (RNA, DNA), such as the RNA obtained in the courseof the genetic transcription of the structural genes contained in thedeoxynucleic acids (DNA) of the cellular organisms from which theyoriginate, there RNA being then capable of being themselves "translated"into proteins capable of being coded by these structural genes. It isknown that these RNA have sequences of nucleotides complementary tothose of the DNA from which they are derived, this complementarity beingmanifested by the capacity possessed by these RNA to form mixed hybridswith corresponding sequences of these DNA previously denatured, inasmuchas the latter were initially bi-catenary, for example after incubationin a high ionic strength medium and at a high temperature or in a basicmedium.

It has been suggested to have recourse, for marking the hybrids formed,to radioactive labeling either of the genes themselves, or of the RNAprobes. These techniques are however difficult to put into practice and,in addition, do not always enable satisfactory localization of the genesconcerned in their DNA.

It is with the object of permitting easier localization of the genesunder study in the DNA containing them, and of promoting a method ofobtaining fractions enriched in predetermined segments of DNA from thesesame DNA that Manning et al. proposed a physico-chemical detectiontechnique for these genes, consisting of chemically modifying the RNAprobe, by fixing biotin groups to the latter, through bridges formed bygroups derived from cytochrome C and fixing physical marks visible withthe scanning (electron) microscope, to the DNA, after hybridization withthe probe, formed by submicroscopic spheres having diameters of about of60 nm, notably based on poly(methacrylate), previously modifiedchemically and coupled in covalent manner to avidin molecules (notablyin the articles entitled "A New Method of in situ Hybridization",Chromosoma (Berl.) 53. 107-117 (1975), Springer-Verlag 1995 and "AMethod for Gene Enrichment Based on the Avidin-Biotin Interaction.Application to the Drosophila Ribosomal RNA Genes", Biochemistry, Vol.16, No. 7, 1364-1369, 1977).

In fact, the incubation of hybrids modified with biotin in the presenceof submicroscopic spheres modified with avidin permits the "labeling"and makes locatable the positions of the desired genes in the DNA whichcontains them, with respect to the overall structure also visible in theelectronic microscope of this DNA, due to the fact of the very powerfulnon-covalent interactions which are then produced between the sitesremaining free of the biotin and of the avidin.

This method however is hardly applicable to the purposes of rapiddetection of the presence or absence of such genes and of such DNA in abiological specimen derived from a human or animal host, for examplewith the object of establishing rapid diagnosis either of the diseasewith which the host may possibly be afflicted, or of the integrity ornot of a gene or of a DNA sequence, for example, in this host.

The invention arises from the conversion of the Manning et al. method,which conversion leads to techniques of detection, even ofcharacterization, capable of being applied in the absence of expensiveequipment, by persons having only little laboratory experience.

The method of detection according to the invention of the possiblepresence or of the characterization of a sequence or particular fragmentof nucleic acid, notably of a gene, even of the whole nucleic acid in acomplex sample of nucleic acids, by contacting the sample, if necessaryafter prior denaturation of the nucleic acid under study, with a probecomprising a complementary nucleic acid, capable of being hybridizedwith the nucleic acid sequence or the nucleic acid sought, ischaracterized in that the reagent or probe used is a probe modifiedchemically by coupling or for its coupling with an enzyme prior orsubsequent to the hybridization reaction, the possible presence ofnucleic acid sequence or of the nucleic acid sought being revealable bythe action of the thus-transformed hybridization product of the probeand of the sequence or of the nucleic acid sought, on an enzymesubstrate.

Advantageously, the enzyme is selected according to its capacity to acton a chromogen substrate, which permits the measurement by optical orsimilar analysis, of the conversion ratio of the substrate, which ratiois then correlatable with the presence or not of the nucleic acidsequence or of the nucleic acid sought in the initial sample.

In a preferred embodiment of the application of the invention, the probeis modified by a chemical group capable of forming a stable complex withthe enzyme or a molecule itself bound stably to the enzyme.Advantageously, the above-said chemical group and the above-saidmolecule are respectively constituted by biotin and avidin or viceversa, the enzyme being itself advantageously constituted byβ-galactosidase.

It is self-evident that the chemical modification must be such that itdoes not prevent the possible subsequent hybridization of the probe withthe DNA sequence or fragment sought.

It will immediately appear to the specialist that this technique permitsa rapid determination of the presence or not in a biological sample ofthe gene or DNA fragment corresponding to the probe used, and this evenin the presence of a considerable amount of other nucleic acids. This isparticularly so due to the fact of the amplification at the level ofdetection which is obtained by the action of the enzyme fixed to thehybrid on the substrate brought into its presence. It is even possible,after sufficient purification of the hybrid, to obtain an indication asto the concentration in the DNA sought in the biological studied or asto the distribution ratio of the gene sought in a purified DNA, bymeasurement of the enzymatic activity observed.

Starting from a nucleic acid sample to be studied, it is possible tofirst carry out the hybridization, then the coupling reaction betweenthe chemically modified and hybridized probe, on the one hand, of theenzyme, on the other hand, to then proceed with the separation or thedegradation of the possible excess of non-hybridized probe and of theexcess enzyme which has not reacted with the probe, before carrying outthe above mentioned measurement.

As an alternative, the separation or degradation of the possible excessof non-hybridized probe may be carried out before the coupling, reactionbetween the chemically modified and hybridized probe, on the one hand,and the enzyme, on the other hand.

The specific probe can be constituted by any specific RNA or DNA eithersingle strand (mono-catenary), or denaturated previously by techniquesknown per se, if it relates to a DNA (or an RNA) initially doublestrand.

When the chemical modification of the probe is carried out by means ofbiotin, it is possible to resort to the technique described by Manninget al. in the already mentioned publications, through cytochrome C,notably in the proportion of one molecule of biotin on the average forabout 100 nucleotides.

Advantageously, recourse is then had for labeling the hybrid by theenzyme, to the product resulting from the coupling of avidin and theenzyme, notably β-galactosidase, by the Avrameas method("Immunochemistry", 1969, 6, 43-52).

It goes without saying that it is possible to resort to other chemicalmodifications of the probe and, if necessary, of the enzyme, to effecttheir coupling, preferably after the hybridization reaction, and that itis possible to reverse the modifying agents of the probe acid of theenzyme respectively.

Other pairs of modifying agents of the probe on the one hand, and of theenzyme, on the other hand, may also be used. By way of example, thefollowing pairs are mentioned, the first of these agents beingpreferably used for the chemical modification of the probe and thesecond for the chemical modification of the enzyme. For example, theprobe may be modified, by a known method, by metallic ions (mercury forexample) and the development is done by means of an enzyme havinghydrosulphide groups (--SH), or coupled to a support including suchgroups.

By way of example, which is of course non-limiting, of an experimentalprocedure which may be applied in the case where the sample to beanalyzed is constituted by a blood specimen of some milliliters, it ispossible to operate as follows:

The blood cells are first lysed and the DNA is extracted therefrom by aconventional technique.

A small amount of the DNA obtained, for example comprised between 1 and100 μg, is denatured by 0.1 to 0.3 N soda, the solution then beingneutralized and brought back to pH 7.

To the solution obtained, the probe corresponding to the DNA fragment orto the DNA sought is then added in the proportion of about 1 μg of probeper 100 μg of denatured DNA (the amount of soda to be used is a functionof the proportion of DNA sought in the specimen to be analyzed). Thesolution is then completed with salts for conferring on the medium ahigh ionic force, at least 0.3 M NaCl, in the presence of 50% formamideand a chelating agent at low concentration, preferably in small volume.The hybridization can then be carried out at ordinary temperature for 1to 40 hours (generally overnight). It is also possible to use thetechnique already described by Manning. As an alternative, any otherhybridization technique can also be resorted to, for example, thatdescribed by KOHNE et al "Biochemistry" (1977) (16, 5329-5341), atordinary temperature in a phenol emulsion.

Avidin coupled to an enzyme such as β-galactosidase is then added to themedium under conditions permitting the coupling of the biotin of theprobe with the free groups of the avidin of the coupling compound of theavidin and the enzyme.

The non-hybridized reagent is then separated from the hybridized reagentby conventional techniques, such as precipitation with polyethyleneglycol, passage over gel, for example that of the type named SEPHAROSE,ultra-centrifugation, etc.

As an alternative it is also possible to carry out the separation of thenon-hybridized probe before the coupling of the avidin bearing theenzyme with the biotin groups coupled to the hybridized probe with theDNA.

The enzyme possibly fixed and consequently the possible effectivehybridization of the probe with the DNA studied may be visualized ordetected by placing in contact with the medium a substrate of theenzyme, notably that constituted by orthonitrophenol galactoside (ONPG).

It is self-evident that the experimental conditions once well-fixed, itis possible to determine a measurable activity threshold, for example,by a colorimetric or fluorographic technique, beyond which it ispossible to conclude in the presence in the treated sample of DNA or ofthe DNA fragments sought.

The following description of a test carried out in the laboratory hassimply the purpose of illustrating the manner in which the processaccording to the invention may be put into practice, it being obviouslyunderstood that the modifications at the level of techniques, accordingto the nature of the biological specimen studied and of that of the DNAor of the DNA fragment, sought, are within the evident scope of thetechnician skilled in the art.

Experiments were carried out on the model consisting of detecting thepresence of a mouse DNA by hybridization of this DNA with a mouseribosomic RNA used as a probe.

Mouse DNA (100 μg per 100 μl of aqueous solution) is denatured byaddition of soda (10 μl of 1 M NaOH). 10 minutes later, the solution wasbrought back to pH neutral by the addition of 10 μl of 1.5 M acid sodiumphosphate NaH₂ PO₄.

1 μg of ribosomic RNA labeled with biotin by means of cytochrome C,prepared by the technique of Manning et al., is added to the denaturedDNA solution. The volume was adjusted to 160 μl with water. 40 μl of asolution having a concentration of mineral salts equal to twenty timesthat of the solution called SSC (abbreviation of the English expression"standard saline citrate") and 200 μl of redistilled or deionizedformamide was then added to the medium. It is recalled that the SSCsolution is an aqueous solution of 0.15 M sodium chloride, 0.015 Msodium citrate, at pH 7.0.

The mixture was incubated until the next day at ordinary temperature,then dialyzed at 4° C. against a solution having a double concentrationof the SSC solution, then for 8 hours against 500 ml of a phosphatebuffer at pH 7.0 containing phosphate at a concentration of 0.1 M,sodium chloride at a concentration of 1 M andethylenediamine-tetrasodium acetate (EDTA) at a concentration of 0.01 M.The latter dialysis is then repeated twice, each time for 8 hours.

The solution thus-obtained was treated with pancreatic ribonuclease for1 hour at ordinary temperature, to obtain a final concentration of 10 μgper ml of ribonuclease, this treatment permitting the degradation of thenon-hybridized RNA.

To the medium obtained was then added a solution of cytochrome C (1 mgper ml) and 1 microliter of a solution containing 1 mg per ml of avidinand 2 mg per ml of β-galactosidase, of which 1 molecule of 62-galactosidase in seven is coupled with avidin. It is mixed and thesolution is then left to stand at 4° C. for 4 hours. The medium was thendiluted to 10 ml with the phosphate dialysis buffer and the solutionobtained is subjected to ultracentrifugation for 1 hour at 35,000 rpm(in a BECKMAN ROTOR SW 41 centrifuge). The DNA and the hybridized RNAare to be found in the centrifugation culot, as well as the avidinβ-galactosidase bound to this RNA. The supernatant liquor contains thenon-hybridized RNA degraded by the ribonuclease and the unbound avidinβ-galactosidase.

The culot is collected and resuspended in 10 ml of buffer. It isrecentrifuged and the culot is taken up again in 0.5 ml of buffer (tubeNo. 1) and the activity of the β-galactosidase on the ONPG substrate isassayed by the technique described by Miller ("Experiments in bacterialgenetics, 1972, Cold Spring Harbor Laboratory", Cold Spring Harbor,N.Y., U.S.A.), by measurement of the optical density of the medium at420 mp, after incubation of the medium at 37° C. for 30 minutes or more.

Controls are prepared under conditions strictly identical with thosewhich have been described above, except that in a first case the initialaddition of ribosomic RNA (tube No. 2) was omitted and in the other casethe addition of mouse DNA (tube No. 3) was omitted.

The results of the three assays carried out are shown in the tablebelow:

    ______________________________________                      Result of the assay (optical            Contents  density at 420 mu after 30    Tube No. DNA       RNA    minutes at 37° C.)    ______________________________________    1        +         +      0.45    2        +         -      0.14    3        -         +      0.15    ______________________________________

The signs + and -, respectively in the columns under the headings DNAand RNA, signify the presence or absence either of DNA, or of RNA, inthe initial medium.

As can be observed on examining this table, the optical density measuredin tube No. 1 (containing the hybrid) is very significantly greater thanthe optical densities measured in the control tubes.

The experimental model which has just been described thereforeillustrates the conditions under which the possible presence of adesired DNA or DNA fragment may be detected, to the extent that a probecomplementary to this DNA or to this RNA fragment is available byresorting to a simple technique requiring neither very complicatedlaboratory equipment nor a particularly experienced technician.

The invention is applicable particularly advantageously to in vitrodiagnosis operations of the presence, for example in a biological sample(blood sample, specimen of stools, etc.) of various viruses, such asthose named Herpes, Epstein Barr, virus Pox, cytomegalo, etc. In thesame way, the invention may be applied to the diagnosis, for example, ofspecific chromosomic anomalies.

It is also applicable to the realization of bacterial diagnoses, inparticular in the case where individuals are bearers of pathogenicgenes, both expressed and non-expressed (or latent).

It will appear naturally to the specialist, in the case of investigatingan infectious DNA, that it is possible to conclude rapidly as to thehealthy character of the treated biological specimen, and having regardto the nucleic acid or the fragment of nucleic acid sought, in theabsence of induction observe on the chromogenic substrate, or at leastan over-shoot of the activity threshold, either predeterminedexperimentally, or by comparison with controls free of the virus.

Conversely, the absence of action observed with respect to thechromogenic substrate, notably beyond the above-mentioned threshold,can, in the other type of application, envisaged above by way ofexample, translate the presence of an anomaly of the chromosomic anomalysought, in the absence of observed total or partial hybridization,between the probe and the DNA studied.

It is advantageously possible to place, for example, at the disposal ofmedical analysis laboratories, "kits" containing all of the essentialreactants for the application of the process according to the invention.These kits can, in particular, contain a sampling of probescorresponding, for example, to the DNA of the virus or bacteria, ofconventionally sought pathogenic viruses or bacteria, or even of probesrelating to particular genes which should normally be contained inbiological specimens, notably blood specimens, under test.

In this regard, the invention relates hence to a "kit" characterized inthat it comprises:

at least one specific probe formed from RNA or a single RNA strand,characteristic of a nucleic acid sequence or of a nucleic acid to besought, this probe being modified chemically for its coupling with anenzyme,

said enzyme, if necessary, modified so as to be able to be coupled withsaid probe,

a substrate, notably a chromogene, specific to the enzyme,

the reactants necessary for the lysis of the cellular medium to bestudied, notably a blood medium, and for the extraction of nucleic acidsfrom the cells of this medium.

As has already been observed in the foregoing, it is advantageous toconstitute the modified probe by a probe to which biotin is bound, themodified enzyme being then constituted by the enzyme itself, for exampleβ-galactosidase, coupled to avidin.

The invention relates also moreover, by way of novel industrial product,to the coupling product of an enzyme (of which the action may berevealed with respect to a substrate, notably chromogenic) and of aprobe (RNA or single strand DNA), either directly, or through a couplingagent. It relates also again to the coupling product of the enzyme andof at least one chemical molecule, the whole then being capable of beingcoupled in its turn with a probe (RNA or DNA), if necessary modified,itself capable of being hybridized with a DNA or a DNA fragment. By wayof examples of such novel industrial products, may be mentioned thecoupling products of a probe (RNA or DNA) with an enzyme, such asβ-galactosidase, or again coupling products of avidin or of biotin withsuch an enzyme.

Of course, the invention may be applied in other fields of application,notably for the labeling of certain DNA fragments in well-known geneticexperiments seeking to establish the genotype of the DNA concerned. Inparticular, the invention may be applied to the determination of theincorporation or not of a particular DNA fragment in experiments ofgenetic sorting comprising for example operations of transforming DNAfrom an infected cell with a foreign DNA containing the DNA fragmentconcerned or on the contrary operations of transduction including theincorporation of a DNA fragment concerned, normally contained in the DNAof the cell, in the DNA of the virus used for the infection of the cell,etc., to the extent that, of course, a probe constituted by the RNAfragment or DNA complementary to the sought nucleic acid fragment isavailable.

As is self-evident and as emerges already besides from the foregoing,the invention is in no way limited to those of its modes of applicationand embodiments which have been more especially envisaged; itencompasses to the contrary all modifications, notably those whererecourse is had to modifications of the probe which may enable theenzymatic assay of the hybrid and modifications relating to theformation and/or purification of the hybrids, to the labeling or thechemical modification of the DNA studied itself, under conditions whichhave been described above, the RNA probe not being the subject of anyparticular labeling; such an inversion of the reactants may beenvisaged, for example in the case of a DNA including numerous examplesof repetitive genes, that it is desired to isolate from the whole DNA,in the form of a hybrid with a probe, after fragmentation of the DNAconcerned by conventional techniques. It is self-evident that theseequivalents are included within the field of protection defined by theclaims.

By way of yet another modification, it is possible to have recourse to aprocess consisting of marking the hybrid formed by the desired DNA andthe probe, by means of an anti-hybrid antibody, coupled to an enzymesuch as β-galactosidase.

We claim:
 1. A probe for the detection of a nucleic acid containing adetermined nucleic acid sequence in a sample containing other nucleicacids not sought to be detected, wherein said probe contains an enzymecoupled to a nucleic acid sequence complementary to said determinednucleic acid sequence, and said enzyme is capable of exerting ameasurable activity on a substrate specific to the enzyme.
 2. A nucleicacid coupled to an enzyme comprising a coupling agent coupling saidenzyme to said nucleic acid, wherein said nucleic acid coupled to saidenzyme is hybridizable to a nucleic acid sought to be detected in thepresence of nucleic acids not sought to be detected.
 3. A detectionmethod for detecting the presence or absence of a nucleic acid sequencesought to be detected in a sample comprising the steps of:exposing thesample to a nucleic acid sequence that hybridizes to nucleic acidsequence sought to be detected if any of the nucleic acid sequencesought to be detected is contained in the sample to form a hybridizationproduct, wherein said hybridization product includes an enzyme coupledthereto; measuring the activity of said enzyme coupled to saidhybridization product on a substrate specific to said enzyme; anddetecting the presence of any said nucleic acid sequence sought to bedetected.
 4. A method of claim 3, wherein said enzyme is coupled to saidhybridization product through an avidin and biotin complex.
 5. A methodof claim 3, wherein said substrate is a chromogen.
 6. A method of claim4, wherein said substrate is a chromogen.
 7. A detection method fordetecting the presence or absence of a nucleic acid sequence sought tobe detected in a sample by means of a nucleic acid probe comprising anucleic acid, wherein said nucleic acid is hybridizable with saidnucleic acid sequence sought to be detected, comprising:forming ahybridization product if any nucleic acid sought to be detected iscontained in the sample, wherein said hybridization product contains thenucleic acid sought to be detected and said nucleic acid probe; couplingan enzyme to the nucleic acid probe to form an enzyme-hybridizationproduct; measuring the activity of said enzyme-hybridization product ona substrate specific to said enzyme; and detecting the presence of anysaid nucleic acid sought to be detected.
 8. A method of claim 7, whereinsaid enzyme is coupled to said nucleic acid probe through an avidin andbiotin complex.
 9. A method of claim 7, wherein said substrate is achromogen.
 10. A method of claim 8, wherein said substrate is achromogen.
 11. A detection method for detecting the presence or absenceof a determined nucleic acid comprising a determined nucleic acidsequence, said method comprising:forming a hybridization product if anynucleic acid sought to be detected is contained in the sample, whereinsaid hybridization product contains the determined nucleic acid sequenceand a nucleic acid probe that contains nucleic acid complementary tosaid determined nucleic acid sequence; coupling an enzyme to the nucleicacid probe to form an enzyme-hybridization product; measuring theactivity of said enzyme-hybridization product on a substrate specific tosaid enzyme; and detecting the presence of any said determined nucleicacid.
 12. A method of claim 11, wherein said enzyme is coupled to saidhybridization product through an avidin and biotin complex.
 13. A methodof claim 11, wherein said substrate is a chromogen.
 14. A method ofclaim 12, wherein said substrate is a chromogen.
 15. A detection methodfor detecting the presence or absence of a determined nucleic acidcomprising a determined nucleic acid sequence, said methodcomprising:forming a hybridization product if any nucleic acid sought tobe detected is contained in the sample, wherein said hybridizationproduct contains the determined nucleic acid sequence and a nucleic acidprobe that contains nucleic acid complementary to said determinednucleic acid sequence, wherein said nucleic acid probe includes anenzyme coupled thereto; measuring the activity of said enzyme coupled tosaid nucleic acid probe on a substrate specific to said enzyme; anddetecting the presence of any said determined nucleic acid.
 16. A methodof claim 15, wherein said enzyme is coupled to said hybridizationproduct through an avidin and biotin complex.
 17. A method of claim 15,wherein said substrate is a chromogen.
 18. A method of claim 16, whereinsaid substrate is a chromogen.